Tissue preferential codon modified expression cassettes, vectors containing same, and uses thereof

Abstract

Described herein are synonymously altered gene sequences which express protein in differing levels within secretory as compared to non-secretory target tissue. An expression cassette comprising an open reading frame (ORF) for a protein under the control of regulatory sequences which direct expression of the product in cell, which ORF has been modified to preferentially increase expression levels in a selected tissue, wherein the modified ORF is characterized by a triplet frequency of any one of Tables 3-12, 16 or 17.

The invention claimed is: 
     
       1. A method for improving expression of a selected gene product in a selected type of target tissue within a selected species, said method comprising:
 constructing an expression cassette comprising a coding sequence for a selected gene product generated with a selected codon frequency of Table 3, Table 4, Table 5, Table 6, Table 7, Table 8, Table 9, Table 10, Table 11, Table 12, Table 16, or Table 17. 
 
     
     
       2. The method according to  claim 1 , wherein the target tissue is selected from liver, skeletal muscle, and respiratory epithelium. 
     
     
       3. The method according to  claim 1 , wherein the target tissue is skeletal muscle and the selected codon frequency is Table 5, or Table 6. 
     
     
       4. The method according to  claim 1 , wherein the target tissue is liver and the selected codon frequency is Table 9, Table 10, or Table 11. 
     
     
       5. The method according to  claims 1 , wherein the target tissue is respiratory epithelium and the selected codon frequency is Table 16. 
     
     
       6. An expression cassette comprising an open reading frame (ORF) for an antibody construct under the control of regulatory sequences which direct expression of the product in a cell, which ORF has been modified to preferentially increase expression levels in target tissue cells, wherein the modified ORF is characterized by a triplet frequency of any one of Tables 3 to 12. 
     
     
       7. The expression cassette according to  claim 6 , wherein the cell is a liver cell. 
     
     
       8. A vector comprising the expression cassette according to  claim 6 . 
     
     
       9. The vector according to  claim 8 , wherein said vector is a recombinant adeno-associated virus (AAV) having an AAV capsid in which the expression cassette is packaged. 
     
     
       10. The vector according to  claim 9 , wherein the expression cassette comprises AAV inverted terminal repeat (ITR) sequences flanking the modified ORF. 
     
     
       11. The vector according to  claim 10 , wherein the ITRs are from a different AAV than the AAV donor for the capsid. 
     
     
       12. An expression cassette comprising an open reading frame (ORF) for an antibody construct under the control of regulatory sequences which direct expression of the product in cell, which ORF has been modified to preferentially increase expression levels in non-secretory cells, wherein the modified ORF is characterized by a triplet frequency of Table 16 or Table 17. 
     
     
       13. A pharmaceutical composition comprising a vector which comprises one or more expression cassettes of  claim 12 . 
     
     
       14. A composition comprising multiple vectors, wherein each vector comprises an expression cassette of  claim 12 . 
     
     
       15. An expression cassette comprising an open reading frame (ORF) for an antibody construct under the control of regulatory sequences which direct expression of the product in a muscle cell, which ORF has been modified to preferentially increase expression levels in muscle, wherein the modified ORF has a sequence selected from: ORF1 having a sequence of SEQ ID NO: 9, ORF 2 having a sequence of SEQ ID NO: 8, ORF11 having a sequence of SEQ ID NO: 7, ORF26 having a sequence of SEQ ID NO: 6, ORF 28 having a sequence of SEQ ID NO: 5, ORF 30 having a sequence of SEQ ID NO: 4, ORF35 having a sequence of SEQ ID NO: 3, ORF39 having a sequence of SEQ ID NO: 2, ORF40 having a sequence of SEQ ID NO: 1, ORF42 having a sequence of SEQ ID NO: 30, IA having a sequence of SEQ ID NO: 10, or IAM having a sequence of SEQ ID NO: 11. 
     
     
       16. The expression cassette according to  claim 15 , wherein the modified ORF is selected from ORF35 having a sequence of SEQ ID NO: 3 and ORF39 having a sequence of SEQ ID NO: 2. 
     
     
       17. The expression cassette according to  claim 15 , wherein the modified ORF is ORF40 having a sequence of SEQ ID NO: 1. 
     
     
       18. The expression cassette according to  claim 15 , wherein the modified ORF is selected from ORF26 having a sequence of SEQ ID NO: 6 and ORF30 having a sequence of SEQ ID NO: 4. 
     
     
       19. The expression cassette according to  claim 15 , wherein the modified ORF is selected from ORF2 having a sequence of SEQ ID NO: 8, ORF 11 having a sequence of SEQ ID NO: 7, ORF26 having a sequence of SEQ ID NO: 6, ORF35 having a sequence of SEQ ID NO: 3 and IA having a sequence of SEQ ID NO: 10. 
     
     
       20. A packaging host cell in culture comprising the expression cassette according to  claim 15 . 
     
     
       21. An rAAV production system comprising (a) a packaging host cell culture comprising the expression cassette according to  claim 15 , (b) AAV rep sequences, AAV cap sequence, and necessary helper functions required for producing the rAAV. 
     
     
       22. A recombinant AAV comprising an AAV8 capsid and an expression cassette for an anti-HIV antibody, wherein the expression cassette is adapted for expression in a selected target tissue, said expression cassette comprising a modified ORF having a sequence selected from the group consisting of ORF1 having a sequence of SEQ ID NO: 9, ORF 2 having a sequence of SEQ ID NO: 8, ORF11 having a sequence of SEQ ID NO: 7, ORF26 having a sequence of SEQ ID NO: 6, ORF 28 having a sequence of SEQ ID NO: 5, ORF 30 having a sequence of SEQ ID NO: 4, ORF35 having a sequence of SEQ ID NO: 3, ORF39 having a sequence of SEQ ID NO: 2, ORF40 having a sequence of SEQ ID NO: 1, ORF42 having a sequence of SEQ ID NO: 30, IA having a sequence of SEQ ID NO: 10, and IAM having a sequence of SEQ ID NO: 11.